CN111979202A

CN111979202A - Pseudorabies virus attenuated strain and application thereof

Info

Publication number: CN111979202A
Application number: CN202010880377.6A
Authority: CN
Inventors: 单学强; 只勇; 于泽坤; 栾志舫; 张伦; 李思菲; 郝光恩; 杨海明; 杨彦超; 张敏; 马广斌
Original assignee: Qingdao Sinder Pharmaceutical Co ltd; Shandong Sinder Technology Co ltd
Current assignee: Qingdao Sinder Pharmaceutical Co ltd; Shandong Sinder Technology Co ltd
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2020-11-24

Abstract

The invention provides a pseudorabies virus low virulent strain, which is a pseudorabies virus TK deletion strain and is prepared by deleting TK genes from a porcine pseudorabies virus QD strain; the preservation number of the QD strain of the pseudorabies virus is CGMCC No. 10266. The invention also provides the application of the pseudorabies virus low virulent strain in preparing a pseudorabies vaccine. The pseudorabies virus low virulent strain is obtained by performing TK gene deletion treatment on a preserved pseudorabies virus strain, compared with the prior construction method, the method adopts brdU pressure and EGFP fluorescent marker double screening, shortens the purification generation and purification time of the recombinant pseudorabies virus, has low pathogenicity and strong immunogenicity, and can provide effective immune protection for pseudorabies virus susceptible animals such as mice and pigs by using the low virulent strain of the pseudorabies virus.

Description

Pseudorabies virus attenuated strain and application thereof

Technical Field

The invention belongs to the technical field of vaccine preparation, and particularly relates to a pseudorabies virus attenuated strain and application thereof.

Background

The porcine Pseudorabies is an acute infectious disease caused by porcine Pseudorabies virus (PRV), the clinical symptoms of the porcine Pseudorabies are expressed as nervous symptoms of vomiting, lethargy, paralysis and the like of piglets in the lactation period until finally failing and dying, and the death rate is almost 100 percent; the pregnant sows can have symptoms of abortion, dead fetus, mummy fetus and the like after infection; respiratory disease symptoms appear after the fattening pigs are infected; the boar is infected with the traditional Chinese medicine, and symptoms such as testicular swelling, atrophy, semen quality reduction and the like appear; the adult pigs are mostly subjected to recessive infection, and can be detoxified for a long time after being endured, so that the adult pigs become a dangerous infection source. The above characteristics of the pseudorabies cause become one of the serious infectious diseases harming the pig industry all over the world, and great economic loss is brought to the pig industry all over the world. Currently, many european countries have announced the purification of porcine pseudorabies by vaccination combined with serological diagnostic techniques. Since 2011, pseudorabies is heavy in earth.

Research has shown that the pseudorabies virus PRV is a herpes virus (Herpesviridae) with a circular virion, a diameter of 150-180nm, a double-stranded DNA genome and a length of about 145kb and with an envelope. The PRV consists of four parts, namely a length special region (UL region), a short unique region (US region), an internal inverted repeat sequence (IRs region) and a terminal repeat sequence (TRs region) at two sides of the US region. The nucleocapsid is mainly composed of UL19 gene and UL35 gene coding protein, envelope protein exists between the nucleocapsid and the envelope, and at least 14 protein is from virus genome coding. PRV virulence is controlled by a combination of genes, among which are UL10, UL13, UL21, UL23(TK), UL39/40, UL44, UL50 in the UL region, and US3, US7, US8 in the US region.

There is no effective drug for treating pseudorabies, and vaccination is the most direct and effective method for preventing and controlling porcine pseudorabies. The vaccine prepared by the pseudorabies classical strain Bartha strain and the BUK strain is widely applied at present. Bartha K61 is obtained by repeated passages of Bartha strain in heterogenous cells, BUK is obtained by continuous passages of low virulent strain Bucharest strain through chick embryo and chick embryo fibroblast for about 800 generations, and the two strains have certain immune protection effect on pregnant sows and piglets.

The gene deletion vaccine is obtained by removing genes or gene fragments related to virulence through a gene engineering technology, and has the advantages that deletion sites are clear and stable, virulence reflexibility is not easy to occur, the virulence is reduced after deletion, and the immunogenicity is not influenced, but an effective gene deletion vaccine is still lacked in the prevention, control and control of pseudorabies.

Disclosure of Invention

The invention aims to provide a pseudorabies virus attenuated strain and application thereof, thereby making up the defects of the prior art.

The invention firstly provides a pseudorabies virus low virulent strain which is a pseudorabies virus TK deletion strain and is prepared by deleting TK genes from a porcine pseudorabies virus QD strain;

the pseudorabies virus QD strain is a herpes virus I (herpes herpesvirus type I) pseudorabies virus QD strain which is preserved in the general microbiological center of the China Committee for culture Collection of microorganisms of the microbiological research institute of China academy of sciences No. 3 of Navy, No.1 Hozei, 3 Kyoto-Yang district, Beijing, 3 and 6 days in 2015, and the preservation number is CGMCC No. 10266.

The invention also provides the application of the pseudorabies virus low virulent strain in preparing a pseudorabies vaccine.

The pseudorabies vaccine is inoculated to a porcine pseudorabies virus susceptible animal.

The pseudorabies virus susceptible animals are mice and pigs.

The pseudorabies virus low virulent strain is obtained by performing TK gene deletion treatment on a preserved pseudorabies virus strain, compared with the prior construction method, the method adopts brdU pressure and EGFP fluorescent marker double screening, shortens the purification generation and purification time of the recombinant pseudorabies virus, has low pathogenicity and strong immunogenicity, and can provide effective immune protection for pseudorabies virus susceptible animals such as mice and pigs by using the low virulent strain of the pseudorabies virus.

Detailed Description

The materials used in the present invention are described below:

PCDNA3.1-EGFP and pBluescript plasmids are preserved in the laboratory; the virus DNA extraction kit, the gel recovery kit and the plasmid extraction kit are purchased from Omega company; PrimeSTAR polymerase, 2 XGC buffer, dNTP mix, 5000bp maker, pMD18-T kit and Blung destination Ligation are all purchased from Takara bioengineering, Inc.; fetal bovine serum was purchased from jin Yuan kang GmbH; DMEM medium was purchased from Biotechnology engineering, Inc.; brdU is purchased from beijing solibao corporation; low melting point agar powder was purchased from sigma; DH5a competent, Clonexpress Ultra One Step Cloning Kit was purchased from Nanjing Novophilia Biotech, Inc., and the calcium phosphate transfection Kit was purchased from Invitrogen. Mice were purchased from the center of the denmengyue experimental animals; pigs were purchased from Jinfeng laboratory animals Co., Ltd, Jinan.

However, those skilled in the art can select other conventional reagent materials to replace them under the technical idea of the present invention.

The TK gene is located in UL23 region of PRV, full length 963bp, and encodes 320 amino acids, and its main function is to catalyze the phosphorylation of deoxythymidine or pyrimidine to dTTP, and participate in the nucleotide synthesis path to maintain and promote the virus replication.

The present invention will be described in detail with reference to examples.

Example 1: porcine pseudorabies virus strain TK gene amplification and cloning

The method comprises the steps of recovering the pseudorabies virus propagated in PK-15 cells, repeatedly freezing and thawing for 3 times, extracting PRV genome by using a virus DNA extraction kit, carrying out PCR amplification by using the PRV genome as a template, wherein the system is a 50 mu L system, 2 XGC buffer 25 mu L, dNTP mix 4 mu L, primeSTAR enzyme 0.5 mu L, template 1 mu L, upstream primer and downstream primer 1 mu L respectively, and finally supplementing water to 50 mu L. The PCR reaction program comprises pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 1min, annealing at 55 ℃ for 15s, extension at 72 ℃ for 2min, and 30 cycles. After detecting the PCR product by agarose gel electrophoresis with the concentration of 1%, cutting the gel to purify a required target band, and recovering by using an Omega gel recovery kit, wherein the specific process is as follows:

adding equal volume of Binding buffer into the PCR product, melting the adhesive tape at 60 ℃ in a 2mL EP tube, incubating for 7min, and reversing the mixed solution every 2min to ensure that the adhesive tape is fully dissolved. Adding the dissolved solution into Hibind DNA columns of the kit, centrifuging under 10000g/min for 1min without exceeding 700uL, and discarding the solution in the collection tube. After adding 300. mu.L binding buffer again, the mixture was centrifuged. Add SPW buffer 700. mu.L at 10000g/min 1min for centrifugation, repeat this operation 2 times. Then, the PCR product was centrifuged at 13000g/min for 1min, the collection tube was discarded, and the Hibind DNA column was put into a self-contained 1.5mL EP tube, 30uL of precipitation buffer was added, and after standing at room temperature for 2min, the PCR product was collected by centrifugation at 13000g/min for 1 min.

Taking 0.2-20 pmol of recovered PCR product, adding 2 μ L of 10 XBringing kit buffer and 1 μ L of Blringing kit Enzyme Mix, supplementing the mixture with water to 20 μ L of system, reacting at 37 ℃ for 10min, and performing heat treatment at 70 ℃ for 5 min. Then, 5. mu.L of the reaction solution was put into a new microcentrifuge tube, and 1. mu.L of blunt-ended vector DNA (50 ng/. mu.L-100 ng/. mu.L) which had been dephosphorylated was added and mixed well. Then 6. mu.L of Ligation solution I was added and mixed. The reaction was carried out at 16 ℃ for one hour.

The whole reaction mixture was transformed into DH5a E.coli competent cells, spread on ampicillin-resistant LB solid medium plates, and cultured overnight at 37 ℃. When the bacterial strain grows to a proper size, a single colony is picked, the bacterial strain is identified to be positive through PCR, and the plasmid number TKLR-T is extracted and sent to a biological engineering company for sequencing. According to the sequencing result sequence, primers for amplifying the upstream and downstream homologous arms related to the TK gene are designed and synthesized by a biological engineering company.

Example 2: deletion TK gene of QD strain of porcine pseudorabies virus

1. Primer design and PCR amplification

Homologous arm primers TKLP1, TKLP2, TKRP1 and TKRP2 were designed to amplify both sides of the TK gene. Where the PmeI cleavage site (underlined) was introduced, reserved for the EGFP fragment to be added in the middle of the upstream and downstream in subsequent manipulations. Simultaneously, EGFP 1 and EGFP 2 primers for amplifying the EGFP expression cassette are designed for standby. The primers were synthesized by Biotech, Inc., as shown in the following table.

2. Construction of TK transfer vector

Using PKLR-T as a template, the homologous arms TKL and TKR of TK were amplified with TKLP1, TKLP2, TKRP1 and TKRP2, respectively. The PCR system is as follows: 2 XGC buffer 25. mu.L, dNTP mix 4. mu.L, upstream and downstream primers 1. mu.L each, DNA template 1. mu.L, PrimeSTAR enzyme 0.5. mu.L, and finally water to 50. mu.L system. The PCR reaction procedure was: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, and extension at 72 deg.C for 1 min. The PCR product was detected by electrophoresis on a 1% agarose gel. And recovering the PCR product.

And (2) taking pcDNA3.1-EGFP as a template, amplifying the EGFP expression cassette by using primers EGFP 1 and EGFP 2, recovering PCR products after agarose electrophoresis under the same system reaction conditions, connecting the products to a T vector for identification, and then amplifying and recovering the EGFP expression cassette by taking a plasmid with a correct sequence as the template.

The pBluescript vector is digested with EcoRV and recovered, and TK gene deletion plasmid construction is performed according to the instructions of the One Step Cloning Kit of Novozam company, as follows:

the most suitable cloning vector amount [0.02 Xcloning vector base pair ] ng and the appropriate amount of each fragment [0.02 Xcloning vector base pair ] ng were added to a micro reaction tube according to the multi-fragment homologous recombination reaction, and 5. mu.L of 2 XClonExpress Mix was added and made up to 10. mu.L with water. Mixing, incubating at 50 deg.C for 15 min; immediately thereafter, the mixture was cooled on ice. Thawing DH5a on ice, adding 5-10 μ L recombinant product into 100 μ L competent cell, flicking the cell wall, mixing well, and standing on ice for 30 min. Heat shock at 42 deg.c for 45 sec and setting on ice for 2-3 min. 900. mu.L of LB liquid medium was added and shaken at 37 ℃ for 1 hour. Taking 100 mu L of the bacillus subtilis, coating the bacillus subtilis on an LB solid medium plate (containing aminobenzyl resistance), carrying out overnight culture at 37 ℃, selecting a colony to carry out shake culture when the size of the colony is moderate, carrying out PCR identification on the colony to obtain a positive bacterium, extracting a plasmid with a pBluescript-delTK number, sending the plasmid to a biological engineering company for sequencing identification, and preserving the bacterium for later use after the identification is correct.

The pBluescript-delTK plasmid is subjected to enzyme digestion treatment by PmeI, and then is connected with an EGFP expression cassette by the method to construct the pBluescript-delTKEGFP plasmid, and the pBluescript-delTK EGFP plasmid is used for fluorescent labeling and screening after homologous recombination of a first-step deletion TK gene after sequencing is carried out.

3. Construction of Pseudorabies virus delta TK gene strain

Transfection experiments were performed using the Invitrogen calcium phosphate transfection kit. The specific operation method is carried out according to the kit instruction: the co-transfected genome was mixed with the pBluescript-delTKEGFP plasmid in a mass ratio of 6: 4. 8: 2 or 9: 1 (total 10. mu.g) was added to a 1.5mL EP tube, followed by the addition of CaCl₂18 μ L, filled to 150 μ L with culture water and labeled as A-tube. Another 1.5mL EP tube was labeled as tube B, and 150. mu.L of 2 XHBS solution was added to the tube. The solution in tube A was then added dropwise slowly to tube B and incubated at room temperature for 30 min. Dropping the mixed solution into the well of the PK-156 pore plate, treating with 30% water glycerol left shock after 4h, eluting twice before treatment, incubating with water glycerol for 2min, eluting twice, adding 2.5mL culture solution, and culturingAnd (4) observing in the box. When the cells were observed to be diseased using a fluorescence microscope and were green fluorescent, the virus fluid was recovered, indicating that both the transfected viral genome and plasmid had been expressed.

The collected virus liquid is frozen and thawed repeatedly at-80 ℃ for 3 times and then inoculated into a T25 single-layer PK-15 cell bottle, a working concentration of 30mg/L brdU reagent is added into the cell culture liquid for continuous culture, cells in the cell bottle are diseased and have green fluorescence excitation, a homology arm and an EGFP expression cassette are integrated onto a virus genome, the virus liquid is collected and then frozen and thawed repeatedly for 3 times, the cells are inoculated onto a 6-hole plate after being diluted by 100 times, the PK-15 cells cultured by the 6-hole plate single layer are changed into a complete culture solution containing 30mg/L brdU reagent 12h before being inoculated with virus, then virus inoculation and incubation are carried out for 1.5h, elution are carried out, each hole is placed into an incubator at 37 ℃ according to the ratio of 1mL of 2 × DMEM, 0.8mL of 2% low melting point agarose and 0.2mL of serum, and continuous observation is carried out after solidification. When the cells have lesions with proper sizes and green fluorescence, picking corresponding fluorescent plaques, dissolving the corresponding fluorescent plaques in a 500-microliter DMEM culture medium, repeating the purification experiment for 1-2 times, and obtaining the purified pseudorabies virus delta TK-EGFP strain, wherein all the lesions in the wells have the green fluorescence plaques.

Extracting purified DNA genome of the pseudorabies virus delta TK-EGFP strain, co-transfecting the DNA genome with pBluescript-delTK plasmid in PK-15 cells according to the calcium phosphate transfection method, recovering virus liquid after no fluorescent lesion appears, and purifying on a 6-well plate, wherein the purification steps are as above. When all lesion plaques are not subjected to fluorescence excitation, the purified pseudorabies virus delta TK gene strain is obtained.

4. Identification of pseudorabies virus delta TK gene strain and virus TCID₅₀Measurement of (2)

The pseudorabies virus genomic DNA was recovered using the kit, and the DNA fragments were purified using primers TKJDF: cagacccggaagcagaacgg and TKJDR: ctgatgtccccgacgatgaa, performing PCR identification, wherein the upstream and downstream primers are located on both sides of the deleted gene, so that a band cannot be effectively amplified after deletion, a parental strain DNA template is used as a positive control, a PCR product is detected by 1% agarose gel electrophoresis, and the purified recombinant pseudorabies virus genome cannot amplify the TK gene, i.e. the TK gene is proved to be deleted.

Respectively carrying out TCID on the parental pseudorabies virus strain and the PRV-delta TK gene strain on a 96-well plate full of a single-layer PK-15 cell₅₀The measurement of (1). When the PK-15 cells in the 96-well plate grow to be full of a monolayer, the cells after passage are diluted by 10 according to the adaptive toxity ratio^-1—10^-11The cells were seeded in 96-well plates in 1-11 columns, and 100. mu.L of each well was added. The last column was added with an equal volume of cell culture medium as a negative control. Placing 96-well plate at 37 deg.C and 5% CO₂Culturing in an incubator, calculating the number of holes with cytopathic effect after 96h, and calculating the virus TCID according to a Reed-Muench method₅₀The parent strain and the PRV-delta TK gene strain are 10⁹TCID₅₀/mL、10^8.8TCID₅₀and/mL. The TK gene deletion can not influence the virus proliferation on PK-15 cells.

Example 3: pathogenicity of the Pseudorabies Virus DeltaTK Gene Strain

1. Comparison of mouse pathogenicity

The experimental method comprises the following steps: 70 BALB/c mice 6-8 weeks old were selected and randomized into 7 groups of 10 mice each. 1-3 groups of the medicinal composition are injected into the back of a patient with 100 mu L10 subcutaneous injection^6.0TCID₅₀The virus liquid of the delta TK gene-deleted strain was injected subcutaneously into the back of 4-6 groups of patients at 100. mu.L, 10. mu.L^6.0TCID₅₀The remaining group of mice were back-injected with 100 μ L DMEM as a negative control. Mice were observed continuously 14d after inoculation and their mental status, clinical symptoms and death were recorded.

The experimental results are as follows: inoculation with 100. mu.L of 10^6.0TCID₅₀The mice with the Δ TK gene-deleted strain virus fluid showed no clinical symptoms and did not die after 14 days of inoculation. After the parent PRV strain group is inoculated, the symptoms of listlessness, appetite reduction and injection part itching appear after 36 to 48 hours, and all the patients die within 72 to 96 hours. The TK gene is deleted, and pathogenicity and virulence to mice are weakened.

2. Comparison of pig pathogenicity

The experimental method comprises the following steps: selecting 12 PRV pathogen negative and PRV antibody negative 8-9 week-old ternary pigs for pathogenicity comparison test. The method is divided into 3 groups, each group has 4 random heads, the 1 st group is inoculated with parent strains, the 2 nd group is inoculated with delta TK gene deletion strains, and the 3 rd group is a negative control group. The method for counteracting toxic substance comprises dripping 4 mL/head, and separately feeding. Body temperature was measured every day at regular time intervals, and mental status, appetite, desire for drinking, etc. were observed.

The experimental results are as follows: the body temperature begins to rise within 24 hours after the parent strain group attacks the toxin; after 48 hours, the average body temperature is above 41 ℃, appetite and desire are lost, spirit is depressed, and 1 pig shows nervous symptoms; after 72h, 1 pig dies, and the rest 3 pigs show obvious itching and nervous symptoms; after 96h, all of the remaining 3 pigs died. After the delta TK gene deletion strain is subjected to virus attack for 48 hours, the body temperature of all inoculated pigs is increased, and the mental state is satisfactory; all pigs have the body temperature rise within 1.5 ℃ for 72 hours, the mental state is good, and the appetite and drinking desire are normal. The control group 4 pigs were healthy with no clinical symptoms. Test results show that the pathogenicity and toxicity to the pig are weakened after the TK gene is deleted.

Example 4: preparation of vaccine from pseudorabies virus delta TK gene strain

1. Preparation and testing of vaccines

The titer is 10^8.8TCID₅₀The virus liquid of the delta TK gene deletion strain is inactivated by beta propiolactone at 4 ℃ for 48h, grafted on PK-15 monolayer cells at 37 ℃ and 5% CO₂Culturing in an incubator for 48h, and judging that the inactivation test is qualified if no cell lesion exists. Virus fluid was mixed with aqueous adjuvant of sibirak according to 1: 1, mixing and preparing the inactivated vaccine of the delta TK gene deletion strain.

2. Mouse immunogenicity assay

Randomly distributing 20 SPF mice of 5-6 weeks old into 2 groups of 10 mice each, one group having 10 inactivated antigens by intraperitoneal injection^8.8TCID₅₀0.2 mL/mL of delta TK gene deletion strain inactivated vaccine and 0.2 mL/group of intraperitoneal DMEM as negative control. Immunization for 21d followed by 10^6.7TCID₅₀And (3) carrying out virus counteracting on the PRV virulent virus per mL, wherein the virus counteracting amount is 0.1mL per mouse injected subcutaneously, observing the clinical symptoms of the mice after virus counteracting, and counting the death number of the mice.

The experimental results are as follows: after the inactivated vaccine is immunized, the mice in the immunized group have good mental state and normal appetite and drinking, and the mice in the control group haveThere is no difference, which indicates that there is no side effect of vaccine immunization. After the challenge, the control mice began to show obvious clinical symptoms of pseudorabies, listlessness, disordered hair, decreased appetite, itching at the challenge site, gnawing the inoculation site and the like within 72 h. The vaccine immunization group had no clinical symptoms. The continuous observation for 14d shows that the mortality rate of the control group mice is 100 percent, and the mortality rate of the vaccine immunization group is 10 percent, which indicates that the antigen content is 10 percent^8.8TCID₅₀at/mL, the Δ TK gene inactivated vaccine can generate 90% protection rate for SPF mice.

3. Pig immunogenicity assay

10 PRV pathogen negative and PRV antibody negative 14-day-old ternary pigs were selected for this test, and the pigs were randomly divided into two groups of 5 pigs each. Antigen content of first group of intramuscular injections 10^8.8TCID₅₀2 mL/head of a/mL delta TK gene inactivated vaccine, and a second group without vaccine injection, which is used as a blank control group. After immunization, two groups were observed for body temperature, mental state, etc. Sera were collected at 7d, 14d and 21d post immunization for determination of PRV antibodies. Immunization for 21d followed by 10^5.7TCID₅₀The PRV strong poison is attacked in a mode of dripping 4 mL/head, and two groups of clinical symptoms and death conditions after attacking are observed. Collecting dead pigs and tissues and organs such as brain, spleen, lung, inguinal lymph node and the like dissected and killed after 14 days, and observing pathological changes. Collecting blood serum, nose and anus swab 7-14 days after virus challenge, determining PRV virus nucleic acid copy number and separating PRV virus.

The experimental results are as follows: after the inactivated vaccine is injected, the observation is carried out for 14d, the immune group and the control group have good mental state and normal appetite and drinking, the excessive body temperature rise occurs in the immune group for 24-48 h, and then the normal body temperature is recovered. Observing 14d after the challenge, and observing 48h after the control group challenge, wherein the clinical symptoms of body temperature rise and pseudorabies begin to appear, and after 7d, all the pigs in the control group die, and the death rate is 100%; after immune group challenge, the body temperature is increased once at 3 d-5 d, no pig dies after 14d continuous observation, the mental state is good, and the survival rate is 100%. This indicates a re-antigen content of 10^8.8TCID₅₀the/mL delta TK gene inactivated vaccine can generate 100% immune protection for 14-day-old piglets.

The test results show that the delta TK gene inactivated vaccine obtained by the invention has stronger immunogenicity, and can generate better immune protection for both mice and pigs.

Example 5: vaccine efficacy testing

60 SPF mice, 5-6 weeks old, were randomly divided into 12 groups of 5 mice each, of which 3 groups were intraperitoneally injected with 10⁷TCID₅₀Viral fluids of Δ TK gene-deleted strain/mL, 0.1 mL/cell, 3 groups 10⁶TCID₅₀Viral fluids of Δ TK gene-deleted strain/mL, 0.1 mL/cell, 3 groups 10⁵TCID₅₀Virus solutions of Δ TK gene-deleted strain/mL, 0.1 mL/cell, and 3-group DMEM culture solutions as negative controls, and 10 was used at 4d, 7d, and 14d after inoculation, respectively⁶TCID₅₀The strong toxicity of the Chinese herbal medicines is attacked, and each Chinese herbal medicine is 0.1mL per Chinese herbal medicine and is injected at multiple points subcutaneously.

The experimental results are as follows: after the mice are inoculated with the attenuated virus solutions with different concentrations of antigen contents, the mental state is good, the appetite is normal, no clinical symptoms appear, and no difference exists compared with a control group, which indicates that the delta TK gene attenuated virus has no side effect on the mice. 4d after immunization, PRV virulent strains are used for grouping together, and more than 80% of mice in each group of 4d after challenge are dead; 7d post-immunization challenge, 10⁵TCID₅₀The protection rate of/mL group mice is less than 20 percent and 10 percent⁶TCID₅₀The protection rate of mice in the/mL group is 40 percent and 10 percent⁷TCID₅₀The protection rate of the/mL group is 50 percent; 14d post-immunization challenge, 10⁵TCID₅₀The protection rate of mice in the/mL group is 50 percent and 10 percent⁶TCID₅₀The protection rate of mice in the/mL group is 80 percent and 10 percent⁷TCID₅₀The protection rate of the/mL group is 90%. The mortality rate of the mice in the control group is more than 90%. This indicates that 4d after the delta TK gene deletion inactivated vaccine is immunized can not produce effective protection for mice, 7d after the immunization can not achieve ideal protection level for mice, and 10 d after the immunization is carried out for 14d⁶TCID₅₀mL and 10⁷TCID₅₀The two groups/mL can ensure that the mice survive more than 80 percent after PRV strong toxicity attack.

The experimental results show that when the antigen content of the attenuated live vaccine prepared by the delta TK gene deletion strain obtained by the invention reaches 10⁶TCID₅₀At more than mL, immunization14d, can generate better immune protection effect on mice.

Claims

1. The pseudorabies virus low virulent strain is characterized in that the pseudorabies virus low virulent strain is prepared by deleting TK gene from a porcine pseudorabies virus QD strain; the preservation number of the QD strain of the pseudorabies virus is CGMCC No. 10266.

2. Use of the attenuated strain of pseudorabies virus according to claim 1 for the preparation of a vaccine.

3. A vaccine comprising the attenuated strain of pseudorabies virus of claim 1 as an antigen.

4. The vaccine of claim 3, wherein the subject is a pseudorabies virus-susceptible animal.

5. The vaccine of claim 4, wherein said pseudorabies virus susceptible animal is a mouse or a pig.